Nitric oxide (NO) deficiency occurs in CKD irrespective of primary cause and contributes to the high rate of cardiovascular complications and progression of CKD. There are many potential mechanisms for NO deficiency in CKD and this application examines 3 possible pathways: Impaired removal of the endogenous NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA); decreased substrate (L- Arg) availability due to reduction in renal L-Arg synthesis and also reductions in endothelial arginine transport; alterations in the neuronal NOS (nNOS) isoforms in the kidney cortex which predispose to CKD progression. We have preliminary data to support each aim. In vivo studies will involve use of 2 CKD models; 5/6 renal ablation/infarction (A/I) and moderate and severe chronic puromycin aminonucleoside (PAN)-induced kidney damage. The 5/6 AI model will be investigated at mild (=CKD1-2), moderate (=CKD 3) and severe (=CKD 4) stages. Most studies will be in the CKD-vulnerable Sprague-Dawley rat and some in the CKD-resistant Wistar Furth. In the first aim we will investigate how CKD alters renal excretion and metabolism of ADMA as well as hepatic and total ADMA clearance. The specific role of renal ADMA metabolism in normal rats will be determined using kidney selective silencing of each of the two enzymes responsible for ADMA metabolism. In CKD animals, selective renal over-expression of the ADMA metabolizing enzymes will be conducted to determine if this reduces ADMA and retards progression and hypertension development. In Aim 2 we will determine how renal and total (whole body) uptake of citrulline and synthesis of L-Arg differs in CKD vs normal kidneys. We will measure abundance and activity of L-Arg transporters in kidney and vascular endothelium in different CKD models in vitro, and L-Arg uptake in vivo. In the 3rd aim we will test the hypotheses that reduction in the nNOSalpha isoform and/or increases in the nNOSbeta isoform in renal cortex are causally related to the progression of CKD in 2 different models. In each aim we will conduct parallel in vitro measurements of abundance and activity of enzymes of interest. We will also determine how and where oxidative stress develops, and how this impacts on these determinants of NO production. The incidence of CKD is rapidly increasing in the US and these studies will help determine the reasons for the overall NO deficiency of CKD and could lead to new therapeutic approaches.